KAP79 (a.k.a. bovineAKAP75, ratAKAP15O) is a human postsynaptic membrane anchoring protein that binds the cAMP dependent protein kinase (PKA), protein kinase C (PKC) and protein phosphatase 2B, calcineurin (CaN) and is recruited to both NMDA and AMPA glutamate receptors by PSD-95/SAP97 scaffold proteins. AKAP79 membrane/cytoskeletal targeting can be regulated by phosphorylation and Ca2+-calmodulin (CaM) in vitro, and postsynaptic localization of AKAP79/150 can be regulated by glutamate or NMDA in hippocampal neurons. Similar glutamate/NMDA treatments leading to synaptic long-term depression (LTD) have been shown to regulate both PKA phosphorylation and postsynaptic localization of AMPA-type glutamate receptors. Thus, regulation of AKAP79 localization by glutamate receptor signaling could be relevant to mechanisms of synaptic plasticity. The proposed research will further characterize the mechanism of AKAP79 postsynaptic targeting and the role of phosphorylation and Ca2+-CaM in its regulation through three specific aims. Aim 1) Mechanism of AKAP79 postsynaptic membrane targeting: identification of AKAP79 targeting domain binding to PB-Cadherin. The role of AKAP79-cadherin binding in AKAP79 polarized targeting and its regulation by phosphorylation and calmodulin will be explored in vitro and in neurons and epithelial cells. Aim 2) Mapping phosphorylation sites in the AKAP79 targeting domain and interplay between phosphorylation and CaM in regulation of targeting. PKA, PKC and CaMKII phosphorylation sites in the AKAP79 targeting domain will be mapped using site-directed mutagenesis in conjunction with both in vitro and cellular P-32 phosphorylation analysis and assays for regulation of binding to phospholipids and F-actin in vitro and plasma membrane targeting in transfected HEK-293 cells. Aim 3) Regulation of AKAP79/150 postsynaptic membrane localization by glutamate receptor activity in cultured hippocampal neurons and hippocampal slices. In hippocampal neurons, NMDA receptor regulation of AKAP79/150 immuno-localization relative to PSD-95 and SAP97 will be correlated with regulation of AMPA receptor localization. To test the involvement of the AKAP79 signaling complex in regulation of AMPA receptor trafficking, corresponding imaging studies will be done on neurons transfected with AKAP79-GFP wildtype and mutants deficient in PKA binding, PKC binding, CaN binding, CaM binding or targeting domain S/T phosphorylation sites. Finally, regulation of phosphorylation and localization of both AKAP79/ 150 and AMPA receptors will be studied in hippocampal slices treated with NMDA or stimulated electrically to induce LTD or LTP synaptic plasticity.